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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Abstract

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial. 

Introduction

Scanning electron microscope (SEM) ultrastructural analysis and intracellular imaging supplement light microscopy for three-dimensional profiling of prokaryotes, plants, and animals. The high spatial resolution of an SEM makes it one of the most versatile and powerful techniques available for the examination of microstructural characteristics of specimens at the nanometer to micrometer scale. Desiccated specimens are resolved to compositional and topographical structures with intense detail, which provides the foundation for developing valid conclusions about functional relationships1,2,

Protocol

NOTE: Painted turtle (Chrysemys picta) eggs used in this study were collected during the nesting season of May through June 2015-16 from Rice Creek Field Station, Oswego New York with permission obtained from the New York State Department of Environmental Conservation (DEC).

1. Chemical drying method to process embryos for SEM

  1. Collect turtle eggs from field sites during the nesting season. Prepare the incubation chambers in advance, made of plastic boxes with lids (L x W x.......

Representative Results

Figure 1 show scanning electron micrographic analysis of painted turtle (Chrysemys picta) embryos. Painted turtle eggs collected and incubated on a bedding medium, mounted on aluminum stubs following chemical drying were used for SEM imaging (Figure 1A-E). A lateral view of a stage 12 embryo shows the craniofacial structures; maxillary prominence extends beyond the mandibular and limits a well-marked nasal pit medially; five pharyngeal .......

Discussion

In our study, different fixation agents, dehydration and drying methods were tested to prepare three different delicate biological samples for SEM imaging: embryos, eggshells, and fungal cultures. SEM is commonly used for surface analysis, so fixative penetration is less concerning, but it must be understood that poorly fixed internal structures will cause inward shrinking or/and collapsed surface structures. Extended fixation time should also be considered for larger tissue samples, replacing the fixative solution a few.......

Acknowledgements

The authors would like to thank Dr. Daniel Baldassarre, SUNY Oswego for helpful discussions and comments on the manuscript. This study was supported by Rice Creek Associate Grants, Oswego; Challenge Grants SUNY Oswego and National Science Foundation (NSF) Small Grants to PGL and JG.

....

Materials

NameCompanyCatalog NumberComments
AgarFischer ScientificS25127Afor slide cultures
Aluminum pin stubTedpella1611112.7 mm x 8 mm
BD Difco Dehydrated Culture Media: Potato Dextrose AgarBD 213400DF0013-17-6Media for isolation and cultivation of Fungi, yeast and molds
ChloramphenicolFischer BioReagentsBP904-100Antibiotic for media
Coarse VermiculiteGreenhouse MegastoreSO-VER-12bedding medium
Clear 12- well plateCorning07-201-589for fixing embryo
CoverslipsFischer ScientificS17525Bfor slide culture
Critical Point DryerQuorum CPDEMS850critical point drying
Culture dishesFischer Scientific08 747BDISH PETRI 100X10MM 12/PK
EthanolFischer ScientificA406P 4dehydration agent
Forceps- Aquarius TweezersTedpella5804style 4, length 108mm, widh x thickness 0.017 x 0.17 mm
GlutaraldehydeFischer ScientificG151-1fixative
Gold target for sputter coaterDENTON VACUUMTAR001-0158Gold Target, 2.375″ D X .002″
HexamethyldisilazanaFischer ScientificC19479-5000chemical drying agent
Kim wipesKimtechS-8115cleaning
Microscope slidesThermo Scientific67-762-16for slide culture
Microscopy ScissorsTedpella1327Double pointed, stainless steel, 100 mm L (3-5/8").
Micro-scissorsTedpella1346Vannas-type, straight, 80mm L
Moria Perforated Embryo SpoonFine Science Tools10370-17Length 14.5 cm, tip diameter 20 mm, spoon depth 5 mm
Netwell InsertsCorning0330B0915 mm Inserts with 74 µm Mesh Size Polyester Membrane act as handy carriers during specimen processing into different solvents
ParaformaldehydeFischer ScientificT353 500fixative
Peat mossWalmart- Miracle Gro551705263bedding medium
PELCO tabs double stick carbon conductive tapeTedpella500012 mm OD
Sputter coaterDENTON VACUUMDESK Vthin metal coating
SEMJEOL USAJEOL JSM 6610LV scanning electron scopeelectron microscopy

References

  1. Packard, M. J. Ultrastructural Morphology of the Shell and Shell Membrane of Eggs of Common Snapping Turtles (Chelydra serpentina). Journal of Morphology. 165 (2), 187-204 (1980).
  2. Solomon, S. E., Watt, J. M.

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Scanning Electron MicroscopySample PreparationPainted Turtle EmbryoRigid EggshellFungal CultureTissue ProcessingEmbryo CollectionEggshell DissectionFixationDehydrationCritical Point DryingMountingSputter Coating

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